PROJECT SUMMARY Objective. Our long-term goal is to understand the molecular organization of the classical cadherin/?- catnein/?E-catenin complex and its interaction with filamentous (F-)actin, which forms the core structure of epithelial cell-cell contacts, and how it responds to and is regulated by mechanical force. This proposal focuses on how both ?-catenin and force regulate the conformation and binding properties of ?E-catenin. We also aim to understand how clustering of cadherin cell adhesion molecules and mechanical force influence the cellular- scale organization of actin at cell-cell contacts, which is essential for understanding how these structures bear mechanical load. These are important biomedical problems: the cadherin/catenin complex is required for the formation of multicellular tissues, and dysregulation of this complex produces defects in epithelial homeostasis and is associated with metastatic behavior of transformed cells. Rationale. Intercellular adhesion is a defining characteristic of multicellular tissues. The F-actin binding protein ?E-catenin and its partner ?-catenin are required to link classical cadherin cell adhesion molecules to the actin cytoskeleton. Surprisingly, the purified ?E-catenin/?-catenin/cadherin complex binds very weakly to F-actin in solution, but we recently found that the strength of the ?E-catenin?F-actin bond increases with mechanical load. Moreover, binding of the cadherin/catenin complex to F-actin is highly cooperative. The combination of these two properties implies that the cadherin/catenin complex may act as a self-reinforcing mechanical linkage between cells whose assembly and disassembly is intrinsically regulated by mechanical force. Clustering of cadherins at cell-cell contacts is another essential property of intact junctions. The proposed experiments aim to uncover how mechanical force regulates the association of ?E-catenin with actin at the molecular level, and how cadherin clustering and tension produce higher-order actin structures at cell-cell contacts. Strategy. We will use state-of-the-art cryo-electron microscopy and spectroscopic methods to investigate the regulated interaction of ?E-catenin with F-actin. In Aims 1 and 2 we investigate how 1) ?-catenin stabilizes a weak F-actin-binding conformation of ?E-catenin, 2) force stabilizes a strong-binding conformation, and 3) tension on the actin filament may promote a conformation of actin that binds strongly to ?E-catenin. In Aim 3, we connect molecular properties with cellular-scale organization by investigating, the effect of cadherin clustering and mechanical tension on the higher-order organization of actin at cell-cell contacts, using cryo- electron tomography in tandem with unique micropatterning methods to apply force to cell pairs.